CN108103102B - AAV1 virus-mediated skeletal muscle specific PCK1 gene expression vector and application thereof - Google Patents

AAV1 virus-mediated skeletal muscle specific PCK1 gene expression vector and application thereof Download PDF

Info

Publication number
CN108103102B
CN108103102B CN201710320647.6A CN201710320647A CN108103102B CN 108103102 B CN108103102 B CN 108103102B CN 201710320647 A CN201710320647 A CN 201710320647A CN 108103102 B CN108103102 B CN 108103102B
Authority
CN
China
Prior art keywords
recombinant
vector
pck1
gene
seq
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN201710320647.6A
Other languages
Chinese (zh)
Other versions
CN108103102A (en
Inventor
田文洪
董小岩
吴小兵
马思思
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Beijing Jinlan Gene Technology Co ltd
Original Assignee
Beijing Jinlan Gene Technology Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Beijing Jinlan Gene Technology Co ltd filed Critical Beijing Jinlan Gene Technology Co ltd
Priority to CN201710320647.6A priority Critical patent/CN108103102B/en
Publication of CN108103102A publication Critical patent/CN108103102A/en
Application granted granted Critical
Publication of CN108103102B publication Critical patent/CN108103102B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
    • C12N15/85—Vectors or expression systems specially adapted for eukaryotic hosts for animal cells
    • C12N15/86—Viral vectors
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K48/00—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy
    • A61K48/005—Medicinal preparations containing genetic material which is inserted into cells of the living body to treat genetic diseases; Gene therapy characterised by an aspect of the 'active' part of the composition delivered, i.e. the nucleic acid delivered
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/10—Transferases (2.)
    • C12N9/12—Transferases (2.) transferring phosphorus containing groups, e.g. kinases (2.7)
    • C12N9/1205—Phosphotransferases with an alcohol group as acceptor (2.7.1), e.g. protein kinases
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y207/00—Transferases transferring phosphorus-containing groups (2.7)
    • C12Y207/01—Phosphotransferases with an alcohol group as acceptor (2.7.1)
    • C12Y207/0104—Pyruvate kinase (2.7.1.40)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2750/00—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA ssDNA viruses
    • C12N2750/00011—Details
    • C12N2750/14011—Parvoviridae
    • C12N2750/14111—Dependovirus, e.g. adenoassociated viruses
    • C12N2750/14141—Use of virus, viral particle or viral elements as a vector
    • C12N2750/14143—Use of virus, viral particle or viral elements as a vector viral genome or elements thereof as genetic vector

Abstract

The invention provides a gene expression and transfer vector for specifically expressing phosphoenolpyruvate carboxykinase (PEPCK) in muscle tissues to reduce hyperlipidemia, delay aging and improve reproductive capacity. The vector contains a promoter of an altered human alpha-skeletal muscle actin (alpha-skeletal actin) gene, an intron of a murine parvovirus (MVM), cDNA of an altered human PCK1 (phosphoenolpyruvatechoxykinase 1, PCK 1) gene and 4 human miR-122 target sequences connected in series. The recombinant vector is mediated by an adeno-associated viral vector, including but not limited to adeno-associated virus type 1.

Description

AAV1 virus-mediated skeletal muscle specific PCK1 gene expression vector and application thereof
Technical Field
The invention relates to the technical field of biology, in particular to an AAV1 virus vector-mediated skeletal muscle specific PCK1 gene expression vector and application thereof, a recombinant expression unit containing the vector and a gene therapy mode.
Background
Phosphoenolpyruvate Carboxykinase (PEPCK) is a rate-limiting enzyme that catalyzes gluconeogenesis (gluconeogenesis) reactions, i.e., the conversion of oxaloacetate and GTP into phosphoenolpyruvate, GDP and CO2The reaction of (1). The Krebs cycle is carried out in mitochondria, while PEPCK has two isoenzymes, PEPCK-M (mitogenic form) in mitochondria and PEPCK-C (cytosolic form) in cytoplasm. The PEPCK-C gene is called PCK1 in human and has an important role in gluconeogenesis. At present, PCK1 becomes the first clear marker of hepatic gluconeogenesis, and the transcription level of the gene in the liver is an important index for clinically evaluating type 2 diabetes, namely if the mRNA expression level of PCK1 is increased, the rate of gluconeogenesis is certain to be increasedAnd (4) increasing.
The PCK1 gene encodes a 63kD protease, and has three main functions: expressed in the liver and renal cortex, catalyzing gluconeogenesis reactions; expressed in the liver and white and brown adipose tissues, involved in the glyceroxenogenic response (gluconeogenesis); plays a role in the loss-of-flow reaction (cataplerosis), removing anions from the tricarboxylic acid cycle so as to greatly increase the kinetics of the tricarboxylic acid cycle. However, the transcriptional activity of PCK1 differs in different tissues. In addition to being predominantly present in the liver, PCK1 is also widely present in mammalian tissues including the small intestine, colon, breast, adrenal gland, lung and muscle, but the metabolic role in these tissues is not yet clear. In addition, the primary function of PCK1 in different tissues is also different, so specific conditional knockdown or overexpression of PCK1 in different tissues will give rise to a variety of different phenotypes. Normally, PCK1 is not expressed or expressed in very low amounts in skeletal muscle. PCK1 uses 6 ATP for its energy to catalyze the conversion of pyruvate to one glucose, whereas in the anaerobic skeletal muscle glycolysis only 2 ATP are produced per glucose. The content of triglycerides in skeletal muscle is proportional to PCK1 and is the main source of energy.
Transgenic mice overexpressing PEPCK-C in muscle (PEPCK-C)mus) Has an exciting phenotype. First, PEPCK-CmusThe transgenic mice have a significantly higher motor capacity than the control mice and run 5 km at 20 m/min, whereas the control mice run only 0.2 km at the same speed, which may be related to an increase in triglycerides in skeletal muscle. Second, PEPCK-CmusTransgenic mice have a much longer lifespan than control mice and produce normal progeny mice at 30-35 months, with most mice becoming incapable of reproduction at 12-18 months (Hakimi P1, Yang J, Casadesus G, et al. overexpression of the cytopathic form of phosphoenolpyruvatechoxykinase (GTP) in skin muscle repattering energy metabolism in the mouse. J. Biol. chem. 2007; 45: 32844. 32855.).
Transgenic pigs overexpressing PEPCK-C in skeletal muscle also gave similar results as mice. The pork with low fat content and high intramuscular fat content, which has similar characteristics with the top-grade beef, is obtained, greatly improves the meat quality and the taste, and has great economic value. And the breeding capability of the PEPCK-C transgenic pig is improved, so that the PEPCK-C transgenic pig has wide application prospect in the breeding of fine breeds.
Adeno-associated virus (AAV) vectors are becoming increasingly important as a new class of safety vectors. It is a parvovirus family member, is a non-enveloped linear single-stranded DNA virus, has a wide host range, can infect both dividing and non-dividing cells, and can mediate long-term expression of foreign genes. As an important member of viral vectors, AAV has no significant cytotoxic effects and does not elicit a cellular strong immune response as other viral vectors do; meanwhile, when the recombinant AAV is constructed, the coding sequence of the AAV is completely deleted, and only the Inverted Terminal Repeat (ITR) with the length of 145 bp at both ends is reserved, so that the possibility of recombination and self-protein expression of the AAV is effectively reduced, and the safety is further improved. Thus, AAV is attracting increasing interest and interest as an ideal gene therapy vector (Lu Y. Recombinant adenovirus-induced virus as delivery vector for gene therapy. Stem Cells Dev. 2004; 13(1): 133-. AAV of different serotypes has slightly different infectivity for different tissues, among which AAV1 has a good infectivity for skeletal muscle (Rebuffat A, et al. Complex of Adeno-Associated Virus Pseudomonas Pseudotype 1, 2, and 8 vector infected by intramucosal infection in the Treatment of human phylogenetic kenonuria.Hum Gene Ther.2010, 21(4), 463 and 477), the target skeletal muscle can be directly injected in multiple points for effective infection.
As described above, under normal physiological conditions, PCK1 is mainly present in the liver, and thus a skeletal muscle-specific PCK1 gene expression vector having high expression and low toxicity can be designed accordingly. Human alpha-skeletal muscle actin is highly expressed in skeletal muscle under normal physiological conditions, so that a promoter of the gene can be selected, and an intron of MVM is added to improve the transcription efficiency so as to realize the specific high expression of PCK1 in the skeletal muscle. And the promoter sequence is subjected to point mutation, and XhoI and SalI enzyme cutting sites in the sequence are eliminated. Then, the cDNA sequence of the PCK1 is connected, and the CDS sequence of the PCK1 gene is subjected to point mutation according to the codon degeneracy principle, so that the EcoRI and BglII enzyme cutting sites are eliminated. Meanwhile, in order to reduce the expression of the exogenously introduced PCK1 gene in the liver, 4 serially connected miRNAs (micro ribonucleic acids) -miR-122 highly expressed in the human liver are introduced after the stop codon of the PCK1 gene, so that the expression of the introduced PCK1 gene in the liver is inhibited, and the possible toxicity caused by the fact that the virus enters the liver through blood circulation to express the PCK1 is too high is avoided.
Therefore, the combination of the promoter of the modified human alpha-skeletal muscle actin gene, the MVM intron, the cDNA of the modified PCK1 gene and the target sequences of 4 tandem miR-122 and the targeted delivery of the recombinant expression vector system of the skeletal muscle by using AAV1 is a good method for realizing the specific expression of PCK1 of the skeletal muscle, and has wide application prospects in treating hyperlipidemia, delaying aging and improving reproductive capacity.
Disclosure of Invention
In view of this, the present invention provides an AAV1 viral vector-mediated skeletal muscle-specific PCK1 gene expression vector and uses thereof, a recombinant expression unit comprising the vector, and a gene therapy method. The recombinant gene expression vector and the virus can effectively reduce the blood fat level so as to achieve the purpose of treating hyperlipidaemia. Meanwhile, the functions of delaying senility and improving reproductive capacity are achieved. The recombinant virus provided by the invention is biologically active and is hopeful to become a candidate virus for treating hyperlipidemia, delaying aging and improving reproductive capacity simultaneously.
In order to achieve the above object, the present invention provides the following technical solutions:
the invention provides a recombinant vector expression unit, which is characterized by comprising:
(1) the nucleotide sequence of the human alpha-skeletal muscle actin gene promoter shown in SEQ ID No. 1; and/or
(2) A nucleotide sequence of a mouse virus MVM intron shown as SEQ ID No. 2; and/or
(3) A cDNA sequence of human PCK1 gene shown as SEQ ID No. 3; and/or
(4) Target sequences of 4 human miR-122 connected in series as shown in SEQ ID No. 4.
The invention also provides a construction method of the recombinant vector expression unit, and the whole base sequence of the recombinant vector expression unit is shown as SEQ ID No. 5.
The present invention provides a full nucleotide sequence of a recombinant vector expression unit, which is characterized by comprising:
(I) a nucleotide sequence shown as SEQ ID No. 5; or
(II) a complementary sequence of the nucleotide sequence shown as SEQ ID No. 5; or
(III) a sequence which encodes the same protein as the nucleotide sequence of (I) or (II) but which differs from the nucleotide sequence of (I) or (II) due to the degeneracy of the genetic code; or
(IV) and a sequence having at least 70% homology with the sequence in (I), (II) or (III).
In some embodiments of the invention, the recombinant vector expression unit is constructed by combining and connecting a promoter of the modified human alpha-skeletal muscle actin gene, an MVM intron, a cDNA of the modified PCK1 gene and target sequences of 4 tandem human miR-122 genes into a vector to construct an expression plasmid vector.
The invention also provides a preparation method of the recombinant plasmid vector and the virus vector, which comprises the following steps:
step 1: the promoter of the modified human alpha-skeletal muscle actin gene, the MVM intron, the cDNA of the modified PCK1 gene and 4 human miR-122 target sequences connected in series are combined and connected to an AAV plasmid vector to construct an expression plasmid vector.
Step 2: and co-transfecting the expression vector and related plasmids into host cells, packaging, harvesting and purifying to obtain the recombinant virus vector.
In some embodiments of the invention, the vector in the method of constructing the recombinant expression vector is a plasmid or a virus.
In some embodiments of the invention, the virus used in the recombinant expression vector construction method includes, but is not limited to, adeno-associated virus type 1.
The recombinant vector expression unit is an AAV1 recombinant expression vector of ITR-alpha-skelteal antibody promoter-MVM intron-PCK1-4 times miR-122T-ITR, which is called HSA-PCK1 for short.
The recombinant vector expression unit comprises: ITR (enhanced-associated virus 2 inverted terminal repeat), alpha-elemental activity promoter, MVM intron (minute virus of minute intron), PCK1 (phosphoenolpyruvate carboxylase 1), 4 × miR-122 target and ITR (enhanced-associated virus 2 inverted terminal repeat).
Wherein the sequence of ITR sequence (Patent WO0220748) is shown as SEQ ID No. 6; the sequence of the modified human alpha-skelestal action protein promoter is shown in SEQ ID No. 1; the sequence of MVM intron is shown in SEQ ID No. 2; the cDNA sequence of the modified human PCK1 gene is shown in SEQ ID No. 3; the sequence of 4 × miR-122 target is shown in SEQ ID No. 4.
The invention also provides a gene therapy mode, which is characterized in that the gene therapy mode is intramuscular multipoint injection of recombinant viruses.
The invention also provides the application of the recombinant expression vector and the gene therapy mode in preparing the medicines for treating hyperlipidemia, delaying aging and improving reproductive capacity.
In the experiment of the invention, a skeletal muscle specific PCK1 recombinant expression vector is designed (figure 1). Under normal physiological conditions, PCK1 is mainly present in the liver, so that a skeletal muscle specific PCK1 gene expression vector with high expression and low toxicity can be designed accordingly. Human alpha-skeletal muscle actin is highly expressed in skeletal muscle under normal physiological conditions, so that a promoter of the gene can be selected, and an intron of MVM is added to improve the transcription efficiency so as to realize the specific high expression of PCK1 in the skeletal muscle. Then, point mutation is carried out on the promoter sequence, and XhoI and SalI enzyme cutting sites in the sequence are eliminated. Then, the cDNA sequence of the PCK1 is connected, and the CDS sequence of the PCK1 gene is subjected to point mutation according to the codon degeneracy principle, so that the EcoRI and BglII enzyme cutting sites are eliminated. Meanwhile, in order to reduce the expression of the PCK1 gene in the liver, 4 serially connected miRNAs with high expression in the liver, namely the base complementary target sequences of miR-122, are introduced behind the stop codon of the PCK1 gene, so that the toxicity possibly caused by the fact that the virus enters the liver through blood circulation to express the PCK1 is too high is avoided.
Further according to the literature (Xiao X, et al, Production of High-Titer Recombinant Adeno-Associated Virus Vectors in the Absence of Helper Adenovir).J Virol.1998, (72) (3): 2224-2232.) the method is used to pack and purify recombinant AAV vector, and the quantitative PCR method is used to determine the genome titer of the virus. Specifically, AAV virus packaging is carried out on the recombinant vector by adopting a three-plasmid cotransfection method, cesium chloride density gradient centrifugation purification packaging is carried out to obtain the recombinant AAV, and the virus genome titer is determined by an SYBR Green quantitative PCR method. AAV viruses of type 1 having a better affinity for skeletal muscle were selected. Injecting AAV1-HSA and AAV1-HSA-PCK1 viruses into a hyperlipidemic mouse animal model in a mode of intramuscular multi-point injection at a dose of 1 × 1011vg/mouse, a significant decrease in total cholesterol and triglyceride levels was observed in the blood of mice in the AAV1-HSA-PCK1 group compared to the control group AAV1-HSA (FIG. 2). The AAV1-HSA-PCK1 is shown to be capable of effectively reducing blood fat and treating hyperlipidemia in vivo.
Mice were further tested for natural senescence to assess the role of AAV1-HSA-PCK1 in delaying senescence. In the survival period of the mouse, the age is early 16-20 months, and the age is 22-24 months. The administration is carried out at 0 month, 6 months and 12 months of mice respectively, and the administration mode is intramuscular multi-point injection, and the injection dose is 1 × 1011 vg/mouse. The results showed that the life span of mice in AAV1-HSA-PCK1 group was significantly prolonged compared to the control group (FIG. 3). The result shows that the AAV1-HSA-PCK1 can effectively delay aging and prolong the life of mice after intramuscular injection at different ages.
The mice were further tested for reproductive ability to assess the role of AAV1-HSA-PCK1 in increasing reproductive ability. Control mice had lost reproductive capacity at 18 months, while mice in the group of AAV1-HSA-PCK1 were still reproductive at 30 months (FIG. 4). The intramuscular injection of AAV1-HSA-PCK1 is shown to effectively improve the reproductive capacity of mice.
The important original experimental materials used in the present invention are as follows:
pHelper plasmid, derived from AAV Helper Free System (Agilent Technologies, USA), was purchased from Agilent Technologies, Inc. and stored. The plasmid contains three plasmids to co-transfect HEK293 cells to prepare adenovirus-derived helper function genes E2A, E4, VA RNA and the like required by recombinant AAV.
The pAAV-R2C1 plasmid was constructed and stored by this company. The pAAV-RC plasmid in AAV Helper Free systems (Agilent Technologies, USA) is used as a basic skeleton, and the sequence from 2013 to 4220 in the pAAV-RC plasmid is replaced by the coat protein coding sequence Cap1 (sequence from 2223 to 4433 in the genome) in AAV1 genome (GenBank ID: NC-002077), so that the pAAV-R2C1 plasmid is obtained. The simple construction process is that pAAV-R2C1 plasmid sequence information is obtained according to the thought, sequences between HindIII and PmeI enzyme cutting sites in the pAAV-R2C1 plasmid are artificially synthesized, and the pAAV-R2C1 plasmid is obtained by replacing the pAAV-RC plasmid with the synthetic sequences by adopting a standard molecular cloning method. The pAAV-R2C1 plasmid contains the cap gene of AAV1 and the Rep gene of AAV2 in a complete form, and 4 Rep proteins (Rep 78, Rep68, Rep52 and Rep 40) and AAV1 coat proteins which are necessary for packaging are provided in the preparation of recombinant AAV1 virus by three-plasmid co-transfection packaging.
The pAAV2neo plasmid is constructed and stored by the company, and is a common AAV plasmid cloning vector which comprises two Inverted Terminal Repeats (ITRs) of AAV2, and elements such as a human cytomegalovirus early promoter, a multiple cloning site and a bovine growth hormone polyA tailing signal are contained between the two ITRs. Plasmid construction procedures are described in the literature (Dong X, et al, assessment of an AAV reverse infection-based array.PLoS ONE2010, 5(10) e13479. Used as a basic backbone for cloning the expression unit of the PCK1 gene in the present invention.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings used in the description of the embodiments or the prior art will be briefly described below.
FIG. 1 shows a design of an expression unit of the recombinant vector of the present invention.
FIG. 2 shows the effect of AAV1-HSA-PCK1 in the treatment of hyperlipidemia in mice; the mice are injected with 1 × 10 dosage by intramuscular injection11vg/AAV 1-HSA and AAV1-HSA-PCK1 virus, mouse blood lipid level.
FIG. 3 shows the effect of AAV1-HSA-PCK1 in delaying senescence in mice; mice were injected intramuscularly at 0 months, 6 months and 12 months with a dose of 1X 1011 vg/AAV 1-HSA and AAV1-HSA-PCK1 virus, mouse longevity extension.
FIG. 4 shows the effect of AAV1-HSA-PCK1 on the enhancement of mouse reproductive performance; the intramuscular injection dosage is 1 × 1011 The mouse reproductive capacity and window phase changed after vg/AAV 1-HSA and AAV1-HSA-PCK 1.
Detailed Description
The invention discloses an AAV1 virus vector-mediated skeletal muscle specific PCK1 gene expression vector and application thereof, a recombinant expression unit containing the vector and a gene therapy mode. The recombinant gene expression vector and the virus can effectively reduce the blood fat level so as to achieve the purpose of treating hyperlipidaemia. Meanwhile, the functions of delaying senility and improving reproductive capacity are achieved. Those skilled in the art can modify the process parameters appropriately to achieve the desired results with reference to the disclosure herein. It is expressly intended that all such similar substitutes and modifications which would be obvious to one skilled in the art are deemed to be included in the invention. While the methods and applications of this invention have been described in terms of preferred embodiments, it will be apparent to those of ordinary skill in the art that variations and modifications in the methods and applications described herein, as well as other suitable variations and combinations, may be made to implement and use the techniques of this invention without departing from the spirit and scope of the invention.
The first purpose of the invention is to provide a recombinant vector expression unit.
The recombinant vector expression unit comprises a promoter of a modified human alpha-skeletal muscle actin (alpha-skeletal actin) gene, an intron of a mouse parvovirus (MVM), a cDNA of a modified human PCK1 (phosphoenolpyruvatecboxykinase 1, PCK 1) gene and 4 miR-122 target sequences connected in series.
The second purpose of the invention is to provide a preparation method of a recombinant vector expression unit, the recombinant vector expression unit structure is an AAV1 recombinant expression vector of ITR-alpha-seelestal action promoter-MVM intron-PCK1-4 XmiR-122T-ITR, which is called HSA-PCK1 for short.
The recombinant vector expression unit comprises: ITR (enhanced-associated virus 2 inverted terminal repeat), alpha-elemental activity promoter, MVM intron (minute virus of minute intron), PCK1 (phosphoenolpyruvate carboxylase 1), 4 × miR-122 target and ITR (enhanced-associated virus 2 inverted terminal repeat).
Wherein the sequence of ITR sequence (Patent WO0220748) is shown as SEQ ID No. 6; the sequence of the modified human alpha-skelestal action protein promoter is shown in SEQ ID No. 1; the sequence of MVM intron is shown in SEQ ID No. 2; the cDNA sequence of the modified human PCK1 gene is shown in SEQ ID No. 3; the sequence of 4 × miR-122 target is shown in SEQ ID No. 4.
The vector is a plasmid or a virus, including but not limited to adeno-associated virus type 1.
The third purpose of the invention is to provide a gene therapy mode, and the gene therapy mode is intramuscular multipoint injection of recombinant viruses.
A fourth object of the present invention is to provide the use of the vector, virus or gene therapy of any of the above in a medicament for treating hyperlipidemia, delaying aging and improving reproductive ability.
In the invention, modern biological technologies and methods such as genetic engineering and the like are adopted to provide a recombinant AAV1 co-expression vector comprising a modified human alpha-skeletal muscle actin gene promoter, an MVM intron, a modified human PCK1 gene cDNA and 4 tandem miR-122 target sequences, and preparation, packaging and application of the virus.
The invention provides an AAV1 virus vector-mediated skeletal muscle specific PCK1 gene expression vector and application thereof, a recombinant expression unit containing the vector and a gene therapy mode. Wherein, unless otherwise specified, the various reagents mentioned in the examples are commercially available; unless otherwise specified, specific procedures described in the examples are described in the third edition of molecular cloning, laboratory Manual.
The invention is further illustrated by the following examples:
EXAMPLE 1 construction of AAV1-HSA-PCK1 recombinant expression vector
The human alpha-skeletal muscle actin promoter sequence was obtained according to literature reports (Mol Cell Biol. 1987; 7(11): 4089-. The MVM intron sequence was then added behind the promoter to give the complete promoter sequence required for expression of the PCK1 gene. This sequence was designated HSA.
Obtaining a cDNA sequence of the PCK1 gene according to a GenBank database, and mutating the obtained PCK1 gene coding sequence according to the codon degeneracy principle to eliminate EcoRI and BglII enzyme cutting sites. Then 4 complete complementary target sequences of miR-122 (highly expressed in liver) are introduced after the stop codon of the PCK1 gene. The sequence of the fragment is named as PCK1-4 times miR-122T.
Two sequences of HSA and PCK1-4 xmiR-122T are synthesized by Nanjing Kinshire biotechnology, and the obtained sequences are cloned into a pUC57 simple vector to obtain pUC57-HSA and pUC57-PCK1-4 xmiR-122T. The plasmid pAAV2neo (Dong X, et al, plasmid of an AAV reverse infection-based array) was used.PLoS ONE2010, 5(10) e 13479.) for cloning backbone, pUC57-HSA vector was first digested with XhoI and KpnI (NEB, USA) to obtain an HSA fragment of 2.3kb in length, which was recovered by agarose gel DNA recovery kit (Tiangen, Beijing, China) after electrophoresis. The pAAV2neo vector was also double-digested with XhoI and KpnI, and recovered by agarose gel DNA recovery kit after electrophoresis. A fragment of the HSA gene andafter the pAAV2neo vector fragments are connected by T4 DNA ligase (Takara, Dalian, China), E. coli DH5 alpha competent cells (Takara, Dalian, China) are transformed, cloning is selected, plasmids are extracted and identified by XhoI and KpnI double enzyme digestion, 2.3kb and 6.9kb fragments are obtained, and the pAAV2neo-HSA vector is obtained. Then, using pAAV2neo-HSA as a basic skeleton, digesting pUC57-PCK1-4 xmiR-122T by KpnI and BglII double enzyme digestion, recovering a fragment with the length of 2058bp, replacing a sequence between KpnI enzyme digestion sites and BglII enzyme digestion sites in the pAAV2neo-HSA vector to obtain a pAAV2neo-HAS-PCK1-4 xmiR-122T vector, and identifying the vector to be correct (6915 bp/2421bp/1118 bp) by XhoI single enzyme digestion to obtain a pAAV2neo-HSA-PCK1-4 xmiR-122T recombinant expression vector, namely HSA-PCK 1.
FIG. 1 shows the recombinant vector expression units designed and constructed.
Example 2 packaging and assay of AAV1-HSA and AAV1-HSA-PCK1 recombinant viruses
Reference is made to the literature (Xiao X, et al, Production of High-Titer Recombinant Adeno-Associated Virus Vectors in the Absence of Helper Adenovir us.J Virol.1998, (72) (2224- & 2232), and packaging and purifying the recombinant AAV using a three plasmid packaging system. Briefly, AAV vector plasmids (pAAV 2neo-HSA or pAAV2neo-HSA-PCK1-4 xmiR-122T), helper plasmids (pHelper) and Rep and Cap protein expression plasmids pAAV-R2C1 of AAV1 are mixed uniformly according to a molar ratio of 1:1:1, HEK293 cells are transfected by a calcium phosphate method, the cells and culture supernatant are harvested after 48h transfection, and the recombinant AAV is separated and purified by a cesium chloride density gradient centrifugation method. Packaging and purifying to obtain 2 recombinant viruses such as AAV1-HSA (packaged by pAAV2neo-HSA plasmid), AAV1-HSA-PCK1 (packaged by pAAV2neo-HAS-PCK1-4 times miR-122T plasmid), and the like.
And determining the genome titer of the prepared AAV by a quantitative PCR method. The specific process is as follows:
two primers, HSA-Q-F and HSA-Q-R, were designed in the HSA promoter:
HSA-Q-F：5’-ATTTTTGGGATGAACTGCCATGATG-3’ (SEQ ID NO.7)
HSA-Q-R：5’-TGGGCCAGAACAGAATCACTCATTT-3’ (SEQ ID NO.8)
by HSA-Q-F and HSA-Q-R are used as primers to specifically amplify an HSA promoter fragment with the length of 177bp, an SYBR Green dye binding method is adopted, 1 mu g/mu l of pAAV2neo-HSA plasmid and a sample diluted by 10 times of gradient are used as standard substances, SYBR Premix Ex Taq II (TliRNaseH Plus) reagent (Takara, Dalian, China) is used, and a fluorescent quantitative PCR instrument (model: ABI 7500 fast, ABI) is used for detecting the virus genome titer. See SYBR Premix Ex Taq II (TliRNaseH Plus) for protocol. Methods for virus treatment are described in the literature (Ulrich-Peter R, et al. Fast and reliable diagnosis of recombinant infected virus type-2 using quantitative real-time PCR).J Virol Methods. 2002; 106: 81-88.）。
Example 3 establishment of hyperlipidemic mouse animal model
12 mice of the C57BL/6J strain are bred at relative humidity of 60 +/-10% and temperature of 22 +/-1 ℃ under the conditions of male and female halves and weight of 18-22 g. After the mice are adaptively fed with the common feed for 3 days, the mice are changed to high-fat feed (corn flour 33.5%, fish meal 5.0%, cholesterol 2.0%, milk powder 4.0%, palm oil 10.0%, soybean 16.7% and other various nutrient substances), and the mice are continuously fed for 16 weeks to successfully establish a hyperlipidemia model. The mice were then subsequently divided into control and experimental groups, each group being male and female. The muscle multi-point injection dose of the control group and the experimental group is 1 multiplied by 1011And 2, continuously feeding the virus Vg/AAV 1-HSA and AAV1-HSA-PCK1 for one month, then taking blood from the eyeball (12 h before blood taking, fasting without water prohibition), centrifuging at 3000rpm for 10min, taking serum, and detecting the content of Total Cholesterol (TC) and Triglyceride (TG) in the serum on a biochemical analyzer.
Total cholesterol and triglyceride changes in blood following AAV1-HSA-PCK1 injection in mice:
FIG. 2 shows that the total cholesterol level and triglyceride level in the blood of mice are significantly reduced compared to the AAV1-HSA control group after the mice are injected with AAV1-HSA-PCK 1.
Example 4 establishment of mouse model of Natural aging and Observation of aging conditions
12 ICR mice are bred at the relative humidity of 60 +/-10% and the temperature of 22 +/-1 ℃ under the conditions that the weight of the mice is 18-22 g and the sex is half. Mice were given a pratAfter 3 days of adaptive feeding by feed, the feed is divided into three groups of control groups and experimental groups, and each group is half male and half female. Mice in control group and experimental group were injected intramuscularly at multiple points at 0 month, 6 months and 12 months, respectively, at a dose of 1X 1011 And (3) continuing feeding the Vg virus and the AAV1 virus and the AAV virus-PCK 1 of the AAV1-HSA and the AAV virus-PCK 1, and observing the aging condition of the mice.
It can be seen that after the AAV1-HSA-PCK1 virus is injected into mice by intramuscular multi-point injection, the survival time of the mice in any age group is obviously prolonged, and the AAV has the effect of delaying senescence.
Survival Change in mice after injection of AAV1-HSA and AAV1-HSA-PCK1 viruses:
FIG. 3 shows that mice of different age groups have the effect of delaying senescence after being injected with AAV1-HSA-PCK1, and the survival time is obviously prolonged as shown in FIG. 3.
Example 5 mouse reproductive Capacity Observation
12 female ICR mice and 2 male ICR mice with the weight of 18-22 g are bred at the relative humidity of 60 +/-10% and the temperature of 22 +/-1 ℃. After 3 days of adaptive feeding of the mice with the common feed, the female mice were immediately divided into a control group and an experimental group, each group consisting of 6 mice. The muscle multi-point injection dose of the control group and the experimental group is 1 multiplied by 1011 The breeding is continued for vg/AAV 1-HSA and AAV1-HSA-PCK1 viruses. Female mice were mated with male mice at 3 months, 18 months and 30 months, respectively, and the reproductive performance of the mice was observed.
After AAV1-HSA-PCK1 injection, the mouse has the following reproductive capacity change:
FIG. 4 shows that after AAV1-HSA-PCK1 is injected into mice, the reproductive capacity of the mice is obviously improved compared with that of an AAV1-HSA control group, and the mice still have the reproductive capacity at 30 months.
The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.
SEQ ID
No.1
5'-CTCGAGAAGGCCCAAATGTAAGCTAGTCCCCTTACGTTACATGCAGCTCATTTGCTAAGTGGTTTTTTTCTAGTATCTCCACTACTCGCTGACACAGGAGGACACAGGATGTTAAAAAGGAAATACAGTTCTGTCAATTATTCACTTACTCTCCAAAATACTTGGAAGAACTAAATATGGAACCATAGGAGACTTTATCCTCACCGCATAGTCCCTATACTAGTCAAACTCCTTATTTTTTAATTGATCATTTTTAGGAAGGTAGCATTTTATTCACTAGAACATTTTTGTTAATACTTGTTTATTTTTGGGATGAACTGCCATGATGTGGGCTACAGAGGAGGGTCGCATATGCTTCCATCCCCCTTTTAGAGAATCCACACCTGTCCCAGTTGCTGGGTTCCACTACCAAAAGTGAATTGCAACTATTTTAGGAGCACTTAAGCACATCCGAAAAATGAGTGATTCTGTTCTGGCCCACACCACATCACTGATGTACCCCCTTAAAGCATGTCCCTGAGTTCATCACAGAAGACTGCTCCTCCTGTGCCCTCCACAAGGTTAGAACTGTCCTTGTCTTAGGGAAAAAGGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGGGACAGGCACCAACTGGGTAACCTCTGCTGACCCCCACTCTACTTTACCATAAGTAGCTCCAAATCCTTCTAGAAAATCTGAAAGGCATAGCCCCATATATCAGTGATATAAATAGAACCTGCAGCAGGCTCTGGTAAATGATGACTACAAGGTGGACTGGGAGGCAGCCCGGCCTTGGCAGGCATCATCCTCTAAATATAAAGATGAGTTTGTTCAGCCTTTGCAGAAGGAAAAACTGCCACCCATCCTAGAGTGCCGCGTCCTTGTCCCCCCACCCCCTCCAATTTATTGGGAGGAAGGACCAGCTAAGCCTCATCTAGGAAGAGCCCCTCACCCATCTCCACCTCCACTCCAGGTCTAGCCAGTCCTGGGTTGTGACCCTTGTCTTTCAGCCCCAGGAGAGGGACACACATAGTGCCACCAAAGAGGCTGGGGGAGGGCCTCAGCCCACCAAAACCTGGGGCCAGTGCGTCCTACAGGAGGGGAACCCTCACCCCTTCAATCCCTTTAGGAGACCCAAGGGCGCTGCGCGTCCCTGAGGCGGACAGCTCCGTGTGCTCAGGCTTTGCGCCTGACAGGCCTATCCCCGGGAGCCCCCGCGCCTCCTCCCCGGCGCTCCGCCCTCGCCTCCCCCCGCCAGTTGTCTATCCTGCGACAGCTGCGCGCCCTCCGGCCGCCGGTGGCCCTCTGTGCGGTGGGGGAAGGGGTTGACGTGGCTCAGCTTTTTGGATTCAGGGAGCTCGGGGGTGGGAAGAGAGAAATGGAGTTCCAGGGGCGTAAAGGAGAGGGAGTTCGCCTTCCTTCCCTTCCTGAGACTCAGGAGTGACTGCTTCTCCAATCCTCCCAAGCCCACCACTCCACACGACTCCCTCTTCCCGGTAGTCGCAAGTGGGAGTTTGGGGATCTGAGCAAAGAACCCGAAGAGGAGTTGAAATATTGGAAGTCAGCAGTCAGGCACCTTCCCGAGCGCCCAGGGCGCTCAGAGTGGACATGGTTGGGGAGGCCTTTGGGACAGGTGCGGTTCCCGGAGCGCAGGCGCACACATGCACCCACCGGCGAACGCGGTGACCCTCGCCCCACCCCATCCCCTCCGGCGGGCAACTGGGTCGGGTCAGGAGGGGCAAACCCGCTAGGGAGACACTCCATATACGGCCCGGCCCGCGTTACCTGGGACCGGGCCAACCCGCTCCTTCTTTGGTCAACGCAGGGGACCCGGGCGGGGGCCCAGGCCGCGAACCGGCCGAGGGAGGGGGCTCTAGTGCCCAACACCCAAATATGGCTTGAGAAGGGCAGCGACATTCCTGCGGGGTGGCGCGGAGGGAATGCCCGCGGGCTATATAAAACCTGAGCAGAGGGACAAGCGGCCACCGCAGCGGACAGCGCCAAGTGAAGCCTCGCTTCCCCTCCGCGGCGACCAGGGCCCGAGCCGAGAGTAGCAGTTGTAGCTACCCGCCCAGGTAGGGCAGGAGTTGGGAGGGGACAGGGGGACAGGGCACTACCGAGGGGAACCTGAAGGACTCCGGGGCAGAACCCAGTCGGTTCACCTGGTCAGCCCCAGGCCTGCGCCCTGAGCGCTGTGCCTCGTCTCCGGAGCCACACGCGCTGGTACC-3'
No.2
5'-GTAAGTTGGCGCCGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTTTTTTACAG-3'
No.3
5'-GGTACCGTAAGTTGGCGCCGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTTTTTTACAGGAATTCGCCACCATGCCTCCTCAGCTGCAAAACGGCCTGAACCTCTCGGCCAAAGTTGTCCAGGGAAGCCTGGACAGCCTACCCCAGGCAGTGAGGGAGTTTCTCGAGAATAACGCTGAGCTGTGTCAGCCTGATCACATCCACATCTGTGACGGCTCTGAGGAGGAGAATGGGCGGCTTCTGGGCCAGATGGAGGAAGAGGGCATCCTCAGGCGGCTGAAGAAGTATGACAACTGCTGGTTGGCTCTCACTGACCCCAGGGATGTGGCCAGGATCGAAAGCAAGACGGTTATCGTCACCCAAGAGCAAAGAGACACAGTGCCCATCCCCAAAACAGGCCTCAGCCAGCTCGGTCGCTGGATGTCAGAGGAGGATTTTGAGAAAGCGTTCAATGCCAGGTTCCCAGGGTGCATGAAAGGTCGCACCATGTACGTCATCCCATTCAGCATGGGGCCGCTGGGCTCGCCTCTGTCAAAGATCGGCATCGAGCTGACGGATTCACCCTACGTGGTGGCCAGCATGCGGATCATGACGCGGATGGGCACGCCCGTCCTGGAAGCAGTGGGCGATGGGGAGTTTGTCAAATGCCTCCATTCTGTGGGGTGCCCTCTGCCTTTACAAAAGCCTTTGGTCAACAACTGGCCCTGCAACCCGGAGCTGACGCTCATCGCCCACCTGCCTGACCGCAGAGAGATCATCTCCTTTGGCAGTGGGTACGGCGGGAACTCGCTGCTCGGGAAGAAGTGCTTTGCTCTCAGGATGGCCAGCCGGCTGGCCAAGGAGGAAGGGTGGCTGGCAGAGCACATGCTGATTCTGGGTATAACCAACCCTGAGGGTGAGAAGAAGTACCTGGCGGCCGCATTTCCCAGCGCCTGCGGGAAGACCAACCTGGCCATGATGAACCCCAGCCTCCCCGGGTGGAAGGTTGAGTGCGTCGGGGATGACATTGCCTGGATGAAGTTTGACGCACAAGGTCATTTAAGGGCCATCAACCCAGAAAATGGCTTTTTCGGTGTCGCTCCTGGGACTTCAGTGAAGACCAACCCCAATGCCATCAAGACCATCCAGAAGAACACAATCTTTACCAATGTGGCCGAGACCAGCGACGGGGGCGTTTACTGGGAAGGCATTGATGAGCCGCTAGCTTCAGGTGTCACCATCACGTCCTGGAAGAATAAGGAGTGGAGCTCAGAGGATGGGGAACCTTGTGCCCACCCCAACTCGAGGTTCTGCACCCCTGCCAGCCAGTGCCCCATCATTGATGCTGCCTGGGAGTCTCCGGAAGGTGTTCCCATTGAAGGCATTATCTTTGGAGGCCGTAGACCTGCTGGTGTCCCTCTAGTCTATGAAGCTCTCAGCTGGCAACATGGAGTCTTTGTGGGGGCGGCCATGAGATCAGAGGCCACAGCGGCTGCAGAACATAAAGGCAAAATCATCATGCATGACCCCTTTGCCATGCGGCCCTTCTTTGGCTACAACTTCGGCAAATACCTGGCCCACTGGCTTAGCATGGCCCAGCACCCAGCAGCCAAACTGCCCAAGATTTTCCATGTCAACTGGTTCCGGAAGGACAAGGAAGGCAAATTCCTCTGGCCAGGCTTTGGAGAGAACTCCAGGGTGCTGGAGTGGATGTTCAACCGGATCGATGGAAAAGCCAGCACCAAGCTCACGCCCATAGGCTACATCCCCAAGGAGGATGCCCTGAACCTGAAAGGCCTGGGGCACATCAACATGATGGAGCTTTTCAGCATCTCCAAGGAGTTCTGGGAGAAGGAGGTGGAAGACATCGAGAAGTATCTGGAGGATCAAGTCAATGCCGACCTCCCCTGTGAAATCGAGAGAGAGATCCTTGCCTTGAAGCAAAGAATAAGCCAGATGT-3'
No.4
5'-AAACACCATTGTCACACTCCAGATCCAAACACCATTGTCACACTCCATAGCCAAACACCATTGTCACACTCCAGATCCAAACACCATTGTCACACTCCA-3'
No.5
5'-CTCGAGAAGGCCCAAATGTAAGCTAGTCCCCTTACGTTACATGCAGCTCATTTGCTAAGTGGTTTTTTTCTAGTATCTCCACTACTCGCTGACACAGGAGGACACAGGATGTTAAAAAGGAAATACAGTTCTGTCAATTATTCACTTACTCTCCAAAATACTTGGAAGAACTAAATATGGAACCATAGGAGACTTTATCCTCACCGCATAGTCCCTATACTAGTCAAACTCCTTATTTTTTAATTGATCATTTTTAGGAAGGTAGCATTTTATTCACTAGAACATTTTTGTTAATACTTGTTTATTTTTGGGATGAACTGCCATGATGTGGGCTACAGAGGAGGGTCGCATATGCTTCCATCCCCCTTTTAGAGAATCCACACCTGTCCCAGTTGCTGGGTTCCACTACCAAAAGTGAATTGCAACTATTTTAGGAGCACTTAAGCACATCCGAAAAATGAGTGATTCTGTTCTGGCCCACACCACATCACTGATGTACCCCCTTAAAGCATGTCCCTGAGTTCATCACAGAAGACTGCTCCTCCTGTGCCCTCCACAAGGTTAGAACTGTCCTTGTCTTAGGGAAAAAGGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGAGGGACAGGCACCAACTGGGTAACCTCTGCTGACCCCCACTCTACTTTACCATAAGTAGCTCCAAATCCTTCTAGAAAATCTGAAAGGCATAGCCCCATATATCAGTGATATAAATAGAACCTGCAGCAGGCTCTGGTAAATGATGACTACAAGGTGGACTGGGAGGCAGCCCGGCCTTGGCAGGCATCATCCTCTAAATATAAAGATGAGTTTGTTCAGCCTTTGCAGAAGGAAAAACTGCCACCCATCCTAGAGTGCCGCGTCCTTGTCCCCCCACCCCCTCCAATTTATTGGGAGGAAGGACCAGCTAAGCCTCATCTAGGAAGAGCCCCTCACCCATCTCCACCTCCACTCCAGGTCTAGCCAGTCCTGGGTTGTGACCCTTGTCTTTCAGCCCCAGGAGAGGGACACACATAGTGCCACCAAAGAGGCTGGGGGAGGGCCTCAGCCCACCAAAACCTGGGGCCAGTGCGTCCTACAGGAGGGGAACCCTCACCCCTTCAATCCCTTTAGGAGACCCAAGGGCGCTGCGCGTCCCTGAGGCGGACAGCTCCGTGTGCTCAGGCTTTGCGCCTGACAGGCCTATCCCCGGGAGCCCCCGCGCCTCCTCCCCGGCGCTCCGCCCTCGCCTCCCCCCGCCAGTTGTCTATCCTGCGACAGCTGCGCGCCCTCCGGCCGCCGGTGGCCCTCTGTGCGGTGGGGGAAGGGGTTGACGTGGCTCAGCTTTTTGGATTCAGGGAGCTCGGGGGTGGGAAGAGAGAAATGGAGTTCCAGGGGCGTAAAGGAGAGGGAGTTCGCCTTCCTTCCCTTCCTGAGACTCAGGAGTGACTGCTTCTCCAATCCTCCCAAGCCCACCACTCCACACGACTCCCTCTTCCCGGTAGTCGCAAGTGGGAGTTTGGGGATCTGAGCAAAGAACCCGAAGAGGAGTTGAAATATTGGAAGTCAGCAGTCAGGCACCTTCCCGAGCGCCCAGGGCGCTCAGAGTGGACATGGTTGGGGAGGCCTTTGGGACAGGTGCGGTTCCCGGAGCGCAGGCGCACACATGCACCCACCGGCGAACGCGGTGACCCTCGCCCCACCCCATCCCCTCCGGCGGGCAACTGGGTCGGGTCAGGAGGGGCAAACCCGCTAGGGAGACACTCCATATACGGCCCGGCCCGCGTTACCTGGGACCGGGCCAACCCGCTCCTTCTTTGGTCAACGCAGGGGACCCGGGCGGGGGCCCAGGCCGCGAACCGGCCGAGGGAGGGGGCTCTAGTGCCCAACACCCAAATATGGCTTGAGAAGGGCAGCGACATTCCTGCGGGGTGGCGCGGAGGGAATGCCCGCGGGCTATATAAAACCTGAGCAGAGGGACAAGCGGCCACCGCAGCGGACAGCGCCAAGTGAAGCCTCGCTTCCCCTCCGCGGCGACCAGGGCCCGAGCCGAGAGTAGCAGTTGTAGCTACCCGCCCAGGTAGGGCAGGAGTTGGGAGGGGACAGGGGGACAGGGCACTACCGAGGGGAACCTGAAGGACTCCGGGGCAGAACCCAGTCGGTTCACCTGGTCAGCCCCAGGCCTGCGCCCTGAGCGCTGTGCCTCGTCTCCGGAGCCACACGCGCTGGTACCGGTACCGTAAGTTGGCGCCGTTTAAGGGATGGTTGGTTGGTGGGGTATTAATGTTTAATTACCTTTTTTACAGGAATTCGCCACCATGCCTCCTCAGCTGCAAAACGGCCTGAACCTCTCGGCCAAAGTTGTCCAGGGAAGCCTGGACAGCCTACCCCAGGCAGTGAGGGAGTTTCTCGAGAATAACGCTGAGCTGTGTCAGCCTGATCACATCCACATCTGTGACGGCTCTGAGGAGGAGAATGGGCGGCTTCTGGGCCAGATGGAGGAAGAGGGCATCCTCAGGCGGCTGAAGAAGTATGACAACTGCTGGTTGGCTCTCACTGACCCCAGGGATGTGGCCAGGATCGAAAGCAAGACGGTTATCGTCACCCAAGAGCAAAGAGACACAGTGCCCATCCCCAAAACAGGCCTCAGCCAGCTCGGTCGCTGGATGTCAGAGGAGGATTTTGAGAAAGCGTTCAATGCCAGGTTCCCAGGGTGCATGAAAGGTCGCACCATGTACGTCATCCCATTCAGCATGGGGCCGCTGGGCTCGCCTCTGTCAAAGATCGGCATCGAGCTGACGGATTCACCCTACGTGGTGGCCAGCATGCGGATCATGACGCGGATGGGCACGCCCGTCCTGGAAGCAGTGGGCGATGGGGAGTTTGTCAAATGCCTCCATTCTGTGGGGTGCCCTCTGCCTTTACAAAAGCCTTTGGTCAACAACTGGCCCTGCAACCCGGAGCTGACGCTCATCGCCCACCTGCCTGACCGCAGAGAGATCATCTCCTTTGGCAGTGGGTACGGCGGGAACTCGCTGCTCGGGAAGAAGTGCTTTGCTCTCAGGATGGCCAGCCGGCTGGCCAAGGAGGAAGGGTGGCTGGCAGAGCACATGCTGATTCTGGGTATAACCAACCCTGAGGGTGAGAAGAAGTACCTGGCGGCCGCATTTCCCAGCGCCTGCGGGAAGACCAACCTGGCCATGATGAACCCCAGCCTCCCCGGGTGGAAGGTTGAGTGCGTCGGGGATGACATTGCCTGGATGAAGTTTGACGCACAAGGTCATTTAAGGGCCATCAACCCAGAAAATGGCTTTTTCGGTGTCGCTCCTGGGACTTCAGTGAAGACCAACCCCAATGCCATCAAGACCATCCAGAAGAACACAATCTTTACCAATGTGGCCGAGACCAGCGACGGGGGCGTTTACTGGGAAGGCATTGATGAGCCGCTAGCTTCAGGTGTCACCATCACGTCCTGGAAGAATAAGGAGTGGAGCTCAGAGGATGGGGAACCTTGTGCCCACCCCAACTCGAGGTTCTGCACCCCTGCCAGCCAGTGCCCCATCATTGATGCTGCCTGGGAGTCTCCGGAAGGTGTTCCCATTGAAGGCATTATCTTTGGAGGCCGTAGACCTGCTGGTGTCCCTCTAGTCTATGAAGCTCTCAGCTGGCAACATGGAGTCTTTGTGGGGGCGGCCATGAGATCAGAGGCCACAGCGGCTGCAGAACATAAAGGCAAAATCATCATGCATGACCCCTTTGCCATGCGGCCCTTCTTTGGCTACAACTTCGGCAAATACCTGGCCCACTGGCTTAGCATGGCCCAGCACCCAGCAGCCAAACTGCCCAAGATTTTCCATGTCAACTGGTTCCGGAAGGACAAGGAAGGCAAATTCCTCTGGCCAGGCTTTGGAGAGAACTCCAGGGTGCTGGAGTGGATGTTCAACCGGATCGATGGAAAAGCCAGCACCAAGCTCACGCCCATAGGCTACATCCCCAAGGAGGATGCCCTGAACCTGAAAGGCCTGGGGCACATCAACATGATGGAGCTTTTCAGCATCTCCAAGGAGTTCTGGGAGAAGGAGGTGGAAGACATCGAGAAGTATCTGGAGGATCAAGTCAATGCCGACCTCCCCTGTGAAATCGAGAGAGAGATCCTTGCCTTGAAGCAAAGAATAAGCCAGATGTGATAAGTCGACAAACACCATTGTCACACTCCAGATCCAAACACCATTGTCACACTCCATAGCCAAACACCATTGTCACACTCCAGATCCAAACACCATTGTCACACTCCAAGATCT-3'
No.6
5'-GACGGCGCTAGGATCATCAACGAAACCCAGCATCTACACAATGTAGCTCAAGTATTCTGGTCACAGAATACAACGAAACCCAGCATCTACACAATGTAGCTCAAGATGATCCTAGCGCCGTCTT-3'
No.7
5'-ATTTTTGGGATGAACTGCCATGATG-3'
No.8
5'-TGGGCCAGAACAGAATCACTCATTT-3'

Claims (8)

1. A recombinant vector expression unit comprising:
(1) the nucleotide sequence of the human alpha-skeletal muscle actin gene promoter shown in SEQ ID No. 1;
(2) a nucleotide sequence of a mouse virus MVM intron shown as SEQ ID No. 2;
(3) a cDNA sequence of human PCK1 gene shown as SEQ ID No. 3; and
(4) 4 human miR-122 target sequences connected in series as shown in SEQ ID No. 4.
2. The recombinant vector expression unit according to claim 1, wherein the entire nucleotide sequence is shown in SEQ ID No. 5.
3. A recombinant vector expression unit comprising:
(I) a nucleotide sequence shown as SEQ ID No. 5; or
(II) a complementary sequence of the nucleotide sequence shown as SEQ ID No. 5; or
(III) a sequence which encodes the same protein as the nucleotide sequence of (I) or (II) but which differs from the nucleotide sequence of (I) or (II) due to the degeneracy of the genetic code.
4. The recombinant vector expression unit according to any one of claims 1 to 3, wherein the expression unit is comprised in a recombinant plasmid vector or in a recombinant viral vector.
5. The recombinant vector expression unit of claim 4, wherein the recombinant viral vector comprises a recombinant adeno-associated virus type 1 vector.
6. A gene therapeutic agent comprising the recombinant vector expression unit according to any one of claims 1 to 5.
7. The gene therapeutic agent according to claim 6, wherein the gene therapeutic agent is a recombinant viral vector, and the recombinant viral vector is a recombinant viral vector for intramuscular multipoint injection.
8. Use of the recombinant vector expression unit according to any one of claims 1-5 or the gene therapy agent according to any one of claims 6-7 for the preparation of a medicament for reducing hyperlipidemia, delaying aging, and improving reproductive ability.
CN201710320647.6A 2017-05-09 2017-05-09 AAV1 virus-mediated skeletal muscle specific PCK1 gene expression vector and application thereof Active CN108103102B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201710320647.6A CN108103102B (en) 2017-05-09 2017-05-09 AAV1 virus-mediated skeletal muscle specific PCK1 gene expression vector and application thereof

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201710320647.6A CN108103102B (en) 2017-05-09 2017-05-09 AAV1 virus-mediated skeletal muscle specific PCK1 gene expression vector and application thereof

Publications (2)

Publication Number Publication Date
CN108103102A CN108103102A (en) 2018-06-01
CN108103102B true CN108103102B (en) 2021-04-13

Family

ID=62207035

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201710320647.6A Active CN108103102B (en) 2017-05-09 2017-05-09 AAV1 virus-mediated skeletal muscle specific PCK1 gene expression vector and application thereof

Country Status (1)

Country Link
CN (1) CN108103102B (en)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN108795946B (en) * 2018-06-28 2022-03-04 北京锦篮基因科技有限公司 Recombinant adeno-associated virus carrying SMN1 gene expression cassette and application thereof
CN113337542A (en) * 2021-06-10 2021-09-03 山西大学 Human replication-defective recombinant adenovirus vector for efficiently expressing Leishmania PEPCK

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
DE10066104A1 (en) * 2000-09-08 2003-01-09 Medigene Ag Host cells for packaging recombinant adeno-associated virus (rAAV), process for their preparation and their use
CN102311973A (en) * 2010-07-05 2012-01-11 北京五加和分子医学研究所有限公司 Novel co-expression vector for micro ribonucleic acids (miRNAs) and coding protein genes
CN103074371A (en) * 2011-10-26 2013-05-01 南开大学 Transgenic vector improving bovine lean meat percentage, intramuscular fat content, and reproductive capacity
CN102827874A (en) * 2012-09-18 2012-12-19 中国药科大学 Transgene carrier for changing mouse movement ability and metabolic function by specific expression of PEPCK-C (phosphoenolpyruvate carboxykinase-cytosolic) in skeletal muscle

Also Published As

Publication number Publication date
CN108103102A (en) 2018-06-01

Similar Documents

Publication Publication Date Title
US20210147876A1 (en) Adeno-associated virus variants and methods of use thereof
CN105579465B (en) For the variation AAV and composition, method and purposes in gene transfer to cell, organ and tissue
CA2907799A1 (en) Adeno-associated virus variants and methods of use thereof
EP3236984B1 (en) Adeno-associated virus vectors encoding modified g6pc and uses thereof
CN108368521A (en) GLP-1 and its purposes in the composition for treating metabolic disease
US20220056090A1 (en) Aav-epo for treating companion animals
TW201837173A (en) shRNA expression cassette, polynucleotide sequence carrying same and application thereof sequentially containing a DNA sequence for expressing shRNA and a filling sequence according to a sequence 5'-3'
CN108103102B (en) AAV1 virus-mediated skeletal muscle specific PCK1 gene expression vector and application thereof
CN109762831B (en) Gene drug constructs for the treatment of mucopolysaccharidosis type 3A
CN108611355A (en) The genomic medicine of X-linkage myotublar myopathy
US20220098614A1 (en) Compositions and Methods for Treating Oculopharyngeal Muscular Dystrophy (OPMD)
JP2023505851A (en) Adeno-associated virus vectors for the treatment of Hunter's disease
JP2022530264A (en) Intrathecal and intravenous combination gene therapy for the treatment of infant Batten disease
CN108103058A (en) A kind of gene therapy medicament of type-1 diabetes mellitus
TW202330914A (en) Compositions and methods for in vivo nuclease-mediated treatment of ornithine transcarbamylase (otc) deficiency
CA3219795A1 (en) Recombinant tert-encoding viral genomes and vectors
CN116926047A (en) Functional beta-galactosidase variant, AAV-mediated human beta-galactosidase expression vector and application thereof
EP4222251A1 (en) Human pah expression cassette for treatment of pku by liver-directed gene replacement therapy
WO2023091997A1 (en) Rnai therapy for apbd and lafora disease

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
TA01 Transfer of patent application right

Effective date of registration: 20200813

Address after: 100176 Beijing Daxing District economic and Technological Development Zone, East Road, No. three Jinghai Road 35, high tech Park 2.

Applicant after: Beijing Jinlan Gene Technology Co.,Ltd.

Address before: 100176 Beijing Daxing District economic and Technological Development Zone, East Road, No. three Jinghai Road 35, high tech Park 2.

Applicant before: BEIJING FIVEPLUS MOLECULAR MEDICINE INSTITUTE Co.,Ltd.

TA01 Transfer of patent application right
GR01 Patent grant
GR01 Patent grant